4-nitrotoluene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

Additional evaluation: liver, spleen, kidney, pituitary gland, uterus, uterine cervix and vagina, mammary gland, seminal vesicle and prostate

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene

EC Number:

202-808-0

EC Name:

4-nitrotoluene

Cas Number:

99-99-0

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-4-nitrobenzene

Details on test material:

- Name of test material (as cited in study report): p-nitrotoluene solified-melted
- Analytical purity: 99.8%
-Appearence: greyish congealed mass
- Lot/batch No.: CHN 0462
- Storage condition of test material: room temperature
- Manufacturer: Bayer AG, Germany

Test animals

Species:

rat

Strain:

Wistar

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Polyethylene glycol 400

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

During the two-week mating period each female was paired daily. Females with positive sperm detection were not mated again. females in which insemination had not been detected by the end of this two-week mating period, were mated for another week with the first male of the dose group which had successfully inseminated a female paired with it.

Duration of treatment / exposure:

males: 35 days, females: up to 46 days

Frequency of treatment:

daily

Duration of test:

Exposure period: males: 35 days, females: up to 46 days
Premating exposure period (males): 2 weeks
Premating exposure period (females): 2 weeks
Duration of test: 47 days

No. of animals per sex per dose:

0 mg/kg bw = 12 animals
25 mg/kg bw = 12 animals
100 mg/kg bw = 12 animals
400 mg/kg bw = 7 animals

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: male/female
Duration of test: 46 days

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

25 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

F0-GENERATION
all dosed rats including controls showed severe salivation (probably due to vehicle),
 MORTALITY; 400 mg-dose group:
 3 males and 5 females in the premating period and 1 female sacrificed moribund on day 22 p.c.: ventral posture, hypoactivity, piloerection, intrauterine death of its litter)
FOOD INTAKE, 400mg-group
m/f: severely decreased,
DEVELOPMENT OF BODY WEIGHT GAIN
 25 and 100 mg-groups: body weight gain comparable to controls
400 mg-group, m/f:
 d1-4: -20.8g/-17.6g (control:8.1g/1.3g);
d4-8: 9.5g/-10.4g (control: 8.4g/2.8g);
d8-15: 19.4g/17.0g (control 9.6g/5.5);
400 mg, females body weight gain reduced when compared to controls during
----------gestation (72.2 g versus 111.3g) and
----------lactation (4.0 g versus 27.2 g)
CLINICAL SIGNS OF INTOXICATION
-----400 mg-group:
m/f: piloerection, respiratory sound, sunken flanks, increased water intake and urination, reduced amount of feces
females: hypoactivity, alteration of gait and increased incidence of soft and light colored feces
--      25 mg-group:
 1 female: ventral posture, hypoactivity, high stepping gait, piloerection
 
EFFECTS ON REPRODUCTION(control, low to high dose):
--INSEMINATION INDEX
(no of females inseminated/no of females paired X 100):
100%, 100%, 100%, 100%.
Determination of the time to insemination did not reveal toxicologically relvant effects in comparison to control
values 
--FERTILITY INDEX
(no of females with implantation sites/ no of females inseminated X 100):
75%, 75%, 100%, 100%
--GESTATION INDEX
(no of females with viable pups/no of females with implantation sites X 100): 100%, 100%, 100%, 85.7%
----No of corpora lutea: 21.56, 18.44, 16.42(stat. sign.), 17.67
----No of implantation sites/litter: 13, 12.89, 12.75, 12.33
----Duration of gestation(days): 22.78, 22.44, 22.33, 22.67
PRENATAL LOSS
(Difference between no of implantation sites and the total no of pups littered (living and dead)):
0.89, 1.33, 0.67, 3.17 (stat. sign.)
-
COURSE OF BIRTH:
------400 mg-group: 1 femle was sacrificed of d22 p.c.: all fetuses of its litter were dead
--------25 mg-group: delivering of only 1 pup with a huge hematoma, loss of all other pups on day of birth

F1-PUPS
--LACTATION BEHAVIOUR:
1 pup of the 100 mg- group, 2 pups of the 400 mg-group: no milk ingestion
POSTNATAL DEVELOPMENTof F1 PUPS (control, low to high dose):
-----No pups delivered(mean-no):
12.11, 11.56, 12.08, 9.17
-----No live pups (mean-no,d0/d4):
12.11/12, 11.56/11.25, 11.92/11.33, 9.17/8.33
------Live birth index (%):
100, 100, 98.61, 100
------Viability index (d4 pp, %):
 98.99, 88.89, 95.41, 92.06
-------Sex ratio (d0, % males/liter):
49.94, 48.66, 44.00, 46.43
------Pup clinical observations (frequency/pups/litters, day 0-day 5):---Found dead:     1/1/1; 14/14/1; 5/5/1; 1/1/1
 ---Missing:        0/0/0;  0/0/0;  2/2/1; 4/4/2
---Hypoactivity:   0/0/0;  1/1/1;  0/0/0; 1/1/1
---Hematoma:       0/0/0;  0/0/0;  0/0/0; 5/2/2
 ---Pale skin:      0/0/0;  5/1/1;  1/1/1; 1/1/1
---Tip of tail
---------dark discolored: 0/0/0;  4/1/1;  0/0/0; 0/0/0
---Milk spot not
detectable:     0/0/0;  0/0/0;  1/1/1; 2/2/2
--Tip of tail
missing:        0/0/0;  1/1/1;  0/0/0; 0/0/0

--MEAN PUP WEIGHT:
d0: m/f/total and d4: m/f/total:
-----control-group
6.30/5.96/6.12 and 9.64/9.22/9.43;
-----25 mg-group:
 6.27/5.94/6.07 and 9.42/9.09/9.22;
------100 mg-group:
5.43(p<0.01)/5.28(p<0.5)/5.36(p<0.05) and 8.31(p<0.05)/8.20/8.26;
-----400 mg-group:
4.89(p<0.01)/4.69(p<0.01)/4.80p<0.01) and 6.79(p<0.01)/6.55(p<0.01)/6.68(p<0.01)
 
F0-GENERATION
GROSS PATHOLOGY:
---------mean organ weights
control, low to high dose, abs(g)/rel[%]):
MALE
liver:
 15.23/3.88, 15.52/3.96, 17.51(p<0.05)/4.26(p<0.05), 19.53(p<0.01)/5.04(p<0.01);
spleen:
0.67/0.17, 0.69/0.18, 0.72/0.18, 1.06(p<0.01)/0.28(p<0.01)
kidney weights, testes weight, epididymal weights were comparable to controls
--FEMALE.:
liver and kidney weights were comparable to controls
spleen:
0.56/0.21, 0.598/0.23, 0.59/0.22, 1.07(p<0.01)/0.45(p<0.01)

HISTOPATHOLOGY(MALE; FEMALE):
---------400 mg group:
LIVER:
periportal pigment deposits (2/12 males, 4/12 females),
variable glycogen content (4/12 males, 3/12 females);
KIDNEY:
tubular pigment (3/12 females),
mononuclear infiltration (2/12 females),
tubular vacuolation (5/12 females),
single cell necrosis (2/12 males);
SPLEEN:
congestion (12/12 males, 10/12 females),
increased pigment (2/12 males, 1/12 females)
TESTES:
atrophy, 2/12;
EPIDIDYMIDES:
cellular debris 4/12

----------100 mg- and 25 mg-groups:
no changes attributable to treatment

Applicant's summary and conclusion

Executive summary:

Klaus (2002):

Groups of 12 male and female Wistar rats each were treated daily (by gavage) for two weeks before mating, during the 2 -week mating and one week remating period, during gestation, lactation and up to the day before necropsy with the test substance solved in Polyethylene glycol 400 in doses of 0, 25, 100 and 400 mg/kg bw/day, respectively. Males were necropsied on day 36 of the study and females were necropsied between day 4 to 5 p.p.

F1 pups were sacrified between day 4 to 5 p.p.

Investigations were performed on general tolerance of the test compound by the parenteral animals (including histopatology of testes, epididymes, accessory sexual glands, ovaries, mammae with mamillae, liver, spleen, kidneys, pituitary gland and organs with macroscopic findings) as well as on effects on reproduction including early postnatal development of F1 pups.

The study was performed in 2002 in compliance with international guidelines (OECD No. 421).

Salivation after administration occurred in all study groups and both genders and was most probably based on the vehicle used.

Increased incidence of this finding in dosed groups was most possibly related to an offensive smell and/or gustatory component of the test substance and toxicological relevance was not assumed for this finding.

After start of treatment with the test substance at the 400 mg/kg dose level signs of severe systemic toxicity were observed in both genders including piloerection, respiratory sounds, sunken flanks, increased water intake and urination and reduced amount of feces. In females hypoactivity, alterations of gait and increased incidence of soft and light colored feces were additionally seen. The clinical symptoms occured together with distinctly to severely decreased feed intake and severe body weight loss and resulted in spontaneous death of one male and sacrifice in moribund condition of 2 further males and 5 females up to day 8 of study.

Another female of the 400 mg/kg dose group was sacrificed moribund on day 22 p.c.and revealed intrauterine death of its litter at necropsy (see below).

Recovery was observed thereafter in both genders during second week of the premating period but clinical symptoms, reduced feed intake and reduced body weight gain reappeared in females of the highest dose group during gestation and lactation. Necropsy of the animals of the 400 mg/kg dose group revealed alterations of the gastrointestinal tract in sacrificed/died animals of both genders as well as single cases of alterations of spleen and prenatal litter loss in females.

Increased weight of liver, spleen and to a slight degree of kidney was observed in males of the 400 mg/kg dose group.

Increased weight of kidney and liver were as well observed to a marginal or slight degree in males of the 100 mg/kg dose group. In females only weight of spleen was increased at the 400 mg/kg dose level.

At the lower dose levels treatment relationship could not be completely excluded for reduced feed intake and body weight gain of females during lactation at a dose level of 25 mg/kg and 100 mg/kg bw/day.

Histopathology of fixed organs revealed in both genders morphological evidence of increased turnover of erythrocytes in spleen at a dose level of 100 mg/kg and above and iron pigment and variable glycogen content in the liver at the 400 mg/kg dose level. Histopatological findings of the kidney consisted of single cell necrosis of renal tubular epithelia in males of the 400 mg/kg dose group while lipofuscin pigment was seen in the renal proximal tubuled in females together with vacuolation of tubules (only in sacrificed females). Further on debris was observed in the epididymides at the 400 mg/kg dose level together with exfoliation of spermatids in a single male.

With respect to reproductive parameters including early postnatal development, treatment related effects on course of birth and lactation behaviour could not be completely excluded at the 400 mg/kg dose level. Further on impaired gestation index, increased prenatal loss and reduced litter size at birth occured at the 400 mg/kg dose level together with clinical symptoms (no milk spot, hematomas), impaired pup weight up to day 4 p.p. and impaired viability index up to day 4 p.p.

Pup weight at birth was as well slightly reduced at the 100 mg/kg dose level.

Thus the following LOAEL and NOAEL were determined:

LOAEL maternal toxicity : 25 mg/kg bw/day

NOAEL teratogenicity : 25 mg/kg bw/day